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Splice Site Selection Abrogated in Cancer: Comparison
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Please note this is a comparison between Version 9 by Amina Yu and Version 10 by Amina Yu.

Splicing and alternative splicing (AS) must be tightly regulated, as they have profound effects on gene expression. Various cis-regulatory elements control the fidelity and efficiency of splicing. These include the 5′ and 3′ splice sites (SSs), splicing enhancers, splicing silencers, branch points, and polypyrimidine tracts. A quality control mechanism of splice site selection termed Suppression of Splicing (SOS), was proposed to protect cells from splicing at the numerous intronic unused, latent 5′ splice sites (LSSs) sequences, which are not used under normal growth condition. However, under stress and in cancer thousands of LSSs are activated in splicing resulting in the expression of thousands of aberrant nonsense mRNAs that may be toxic to cells. 

  • pre-mRNA splicing
  • alternative splicing
  • latent splicing
  • aberrant splicing
  • endogenous spliceosome
  • breast cancer
  • glioma

1. Alternative Splicing (AS) Is a Key Regulator of Human Gene Expression

Pre-mRNA splicing is an essential mechanism that controls the inclusion of exons and removal of introns in mature mRNA. Splicing and AS play major roles in regulating gene expression. More than 94% of human genes are estimated to undergo AS [1][2][3][4][5][6], which is considered to be a major source of the diversity of the human proteome. AS has been shown to control almost every aspect of protein function, such as protein localization, enzymatic activity, protein stability, and ligand interaction [6], and thus plays a crucial role in generating tissue and cell-type specific gene expression. Therefore, changes in AS and mis-regulation of AS factors are involved in numerous human diseases including cancer [1][7][8][9][10][11]. Through studies in genetics, molecular biology, and high-resolution cryo-EM, the molecular mechanism of splice site recognition and splicing of pre-mRNAs harboring a single intron is well understood [12][13][14][15][16][17][18][19]. However, it is still not clear how alternative SSs are recognized and selected in pre-mRNAs that contain multiple introns, and currently it is impossible to predict tissue-specific splicing programs.
Approximately 2500 transcription factors are known to regulate ~22,000 genes [20], yet only around 70 sequence-specific splicing regulators have been described [21].Since both AS and promoter recognition are regulated through the combinatorial control of protein factors binding to either DNA or RNA, this scientific challenge prevents a full understanding of human gene regulation.

2. The Endogenous Spliceosome

Nuclear processing of Pol II transcripts, including splicing and AS, takes place in the endogenous spliceosome—the supraspliceosome. This is an enormous (21 MDa) highly dynamic structure comprising four active native spliceosomes joined together by the pre-mRNA [22][23][24][25][26].  The supraspliceosome is an autonomous macromolecular machine, where all nuclear pre-mRNAs, regardless of their length or number of introns, are individually assembled and processed, in a carefully controlled, coordinated fashion. The tetrameric structure of the supraspliceosomes is suitable to coordinate multiple processing events of the pre-mRNA. Accordingly, in previous studies, it was shown that AS is regulated in supraspliceosomes [22][23][27].

3. A Quality Control Mechanism of Splicing Regulation

Recognition and selection of the 5′ SS consensus sequence is a key step in pre-mRNA splicing [3]. Intriguingly, such potential sequences are abundant within human introns [28], but are not used under normal growth conditions, thus termed LSSs. Their use or activation could potentially add intronic sequences containing PTCs into most (98%) alternatively spliced isoforms [28], generating non-functional mRNAs that would be subjected to NMD in the cytoplasm [29][30][31]. Importantly, LSSs are activated under stress and in cancer [28][32][33], resulting in thousands of activated, aberrant gene transcripts [28]. Clearly, maintaining the fidelity of normal splicing is important and tightly controlled, but this can be derailed under certain conditions. Two scenarios might account for the lack of observed splicing at LSSs under normal growth conditions: (i) splicing at LSSs does occur, but an RNA surveillance mechanism, such as NMD [34][35][36], rapidly and efficiently degrades the nonsense mRNAs; (ii) a novel quality control mechanism suppresses splicing at LSSs that are preceded by at least one stop codon in-frame with the upstream exon (Figure 1). Experiments in our lab have ruled out the first scenario of NMD [32][33][37], as well as degradation of latent mRNAs by a yet unknown RNA degradation mechanism [38], while fitting the second scenario of latent splicing suppression. These discoveries led us to suggest a quality control mechanism of pre-mRNA splicing, which was named SOS. This mechanism, which differentiates between normal and LSS, requires recognition of an ORF that enables splicing only at normal 5′SSs, thus avoiding the generation of nonsense RNA transcripts [22][28]. The results show that SOS is an evolutionarily conserved mechanism, likely shared by most eukaryotes [22][33]. Support for a nuclear surveillance mechanism that operates independently of NMD, recognizes PTC-harboring pre-mRNAs in the nucleus and suppresses splicing to prevent the production of such transcripts has been observed in a number of studies [39][40][41][42]. However, the mechanism of the SOS quality control remains nebulous.
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Figure 1. Suppression of splicing (SOS). Two scenarios for lack of latent splicing under normal growth conditions: A scheme depicting the two scenarios that can account for lack of latent splicing under normal growth conditions, despite the abundance of LSS sequences in introns. As discussed, it has been ruled out the first scenario [32][33][37][38]. Boxes, exons; narrow boxes, latent exons; lines, introns; red octagon, stop codon. Adapted from ref. [22].

4. Elements of the SOS Mechanism

4.1. SOS Requires Recognition of the Reading Frame in the Nucleus, Independent of Translation

In an attempt to understand the rules of SOS, the presence of an in-frame stop codon was shown to play a role in suppressing splicing from the latent sites. Specifically, it was demonstrated, through a large series of mutations of gene constructs, that removal of stop codons led to activation of splicing at the latent SSs (latent splicing). These mutations included mutations in the stop codons to produce sense codons, and frame shift mutations by insertion or deletion of nucleotides upstream of the stop codons [32][33][37]. Three different lines of experiments ruled out the possibility that the mutations we made affected splicing through the damage of a splicing control element [43][44] (i) by demonstrating that removal of only one of the two intronic in-frame stop codons present upstream to the LSS in a minigene construct was not enough to produce latent splicing; (ii) by revealing that mutating an in-frame stop codon to the remaining two stop codon sequences did not elicit latent splicing, but mutating the stop codon to a missense codon did; and (iii) by confirming that frame shift mutations, further away from the in-frame stop codon, elicited latent splicing [37].
Splicing control by SOS requires a starting point for the recognition of the mRNA reading frame, and the start codon AUG sequence was a likely candidate. Indeed, using mutagenesis, it was demonstrated that AUG sequences are essential for SOS. Although protein translation does not seem to be required for SOS, the first AUG was shown to be necessary but not sufficient. The abrogation of SOS was attributed to a mutation in the AUG sequence, rather than interference with splicing control elements, because mutating nucleotides in the vicinity of the AUG sequence did not elicit latent splicing [22].

4.2. A Role for Initiator-tRNA in SOS

The finding that mutations in the translation initiation codon, AUG, elicited latent splicing, even though the stop codons remained intact [22], supported the requirements for the conservation of the ORF for the SOS mechanism, with the AUG translation initiation codon as its starting point. This raised the possibility of the initiator-tRNA (ini-tRNA) as a potential SOS factor, through its recognition of the AUG. This was verified by demonstrating that the ini-tRNA has a regulatory role in pre-mRNA splicing, which is not connected with its function in protein translation. It was demonstrated that abrogation of SOS, which occurred upon mutating the AUG translation initiation codon (Figure 2a), can be counterbalanced by ini-tRNA having complementary anticodon mutations to the AUG mutations, thus rescuing SOS. This rescue cannot be achieved by mutated elongator-tRNA [38]. Importantly, the ini-tRNA species that is proposed to participate in SOS resides in the cell nucleus and appears not to be charged with an amino acid. These findings indicate that the nuclear base-pairing of the uncharged ini-tRNA anticodon triplet with the initiation AUG sequence plays a key role in controlling the quality of splicing by the yet undeciphered SOS mechanism [38].
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Figure 2. A potential role for NCL in SOS. The recovery of SOS by ini-tRNA complementation is NCL dependent. (a) Hypothesis and experimental design. Assuming that ini-tRNA is essential in establishing a reading frame required for the SOS mechanism, latent splicing is suppressed when CAD-WT is complemented by ini-tRNA with a complementary anticodon (CAU). It is expected that abrogating SOS in an AUG to ACG mutant (CAD-Mut31), elicits latent splicing, which is rescued by a mutant in-tRNA, which carries a complementary anticodon (CGU) mutation, resulting in a reduced level of latent splicing (CAD-Mut31). These assumptions were verified [38]. Notably, recovery of SOS by ini-tRNA complementation is disrupted by NCL knockdown using siRNA. Scheme: gray box, exon; line, intron; blue box, latent exon; +(w) indicates weak latent splicing. (b,c) Experimental verification using the CAD minigene. HEK 293 cells were co-transfected with CAD-Mut31 (CAD31), which carries the mutated ACG start-codon; with CAD31 together with mutant ini-tRNA, in which the antisense codon was mutated to CGU (ini-CGU), as indicated. Co-transfection of CAD-Mut31 with mutant ini-tRNA and with si-RNA directed against NCL (NCLsi), disrupt the complementation. (c) Graphs represent an average of three independent experiments. The densitometric ratio of CAD31 was normalized to 100%. Adapted from ref. [45].

4.3. A Novel Role for Nucleolin (NCL) in SOS

In the efforts to decipher the SOS mechanism, a search for partners of ini-tRNA in the nucleus was conducted. Starting with UV crosslinking followed by mass spectrometry analysis, NCL was identified as a protein that directly and specifically binds to ini-tRNA in the nucleus but not in the cytoplasm. NCL is an abundant RNA binding protein, with multiple cellular functions (e.g., synthesis and maturation of ribosomes in the nucleolus, a role in Pol II transcription, DNA repair, chromatin decondensation, and genome stability) which are not yet fully understood. Furthermore, NCL is known to play a role in cancer, in which its overexpression affects cell  survival, proliferation, and invasion [46][47]. To establish the relevance of this interaction to SOS, the association of ini-tRNA and NCL together with pre-mRNA was demonstrated. Furthermore, SOS was shown to be NCL-dependent by using a construct mutated in the AUG, thereby abrogating SOS, in combination with a mutant ini-tRNA carrying an anti-codon mutation, which complements the AUG mutations and rescues SOS (Figure 2). Using this system, it was shown that the recovery of SOS by ini-tRNA complementation is NCL-dependent, as the complementation is abrogated by knockdown of NCL (Figure 2b,c). Finally, NCL knockdown resulted in activation of latent splicing in hundreds of coding transcripts that have important cellular functions. Therefore, NCL was proposed as the first protein component in a nuclear quality control mechanism that regulates splice site selection, thereby protecting cells from latent splicing that can generate defective mRNAs [45].

5. SOS is Abrogated under Stress and in Cancer

Previously, it has been shown that heat shock elicited latent splicing in the CAD gene in Syrian hamster cells [28]. Furthermore, heat shock also activated latent splicing in tested C. elegans  transcripts [33].  It was further shown that exposing Syrian hamster cells to γ irradiation, hypoxia, cold shock, and heat shock elicited latent splicing in endogenous CAD mRNA, with heat shock causing the strongest effect [28]. Therefore, the global effect of heat shock on latent splicing was examined using a splicing-sensitive microarray, revealing activation of splicing in 508 latent sites. It should be pointed out that this number of activated LSSs is a lower limit, because the latent transcript, which contains PTCs, is downregulated by NMD in the cytoplasm [28].

Importantly, SOS is abrogated in cancer, resulting in activation of thousands of LSSs. For female breast cancer, the second most commonly diagnosed and the fifth leading cause of cancer-related deaths worldwide [48][49][50][51][52], ， data mined from the Gene Expression Omnibus (GEO) of MCF-7 breast cancer (BC) cells as compared to MCF-10A non-malignant breast cells [53] was analyzed [28]. The analysis revealed activation of latent splicing in 794 latent sites [28]. Brain cancers are characterized by high morbidity and mortality, owing to their localization and often local invasive growth [54][55]. Gliomas are the most common primary central nervous system tumors in adults, and despite advances in treatments, the prognosis for most  glioma patients remains poor [55].  Similar analyses of activation of latent splicing were performed in different types of gliomas, using data available in the GEO database [56]. This analysis revealed that in glioblastoma tumors, 409 latent sites were activated, while in oligodendroglioma (OD) samples the number of activated LSSs were 853 in grade II and 612 in grade III [28]. These mRNAs are from broad functional groups, including mRNAs implicated in cell differentiation and proliferation. Notably, in OD, a correlation was found between the level of activation of latent splicing and the severity of the disease. The analysis revealed 125 mRNAs for which the extent of latent splicing activation was higher in the more aggressive ODIII than in ODII or normal cells (Figure 3), portraying novel markers for OD.

Figure 3. Activation of latent splicing in cancer: Correlation between the level of activation of latent splicing and the severity of OD. The graph depicts fold changes in the level of latent splicing in 125 gene transcripts whose latent splicing expression increased from normal cells to ODII and further increased in ODIII. Names of the top-scoring gene transcripts are indicated [28].

Interesting examples are genes expressing aberrant nonsense mRNAs in both breast cancer and glioma, due to latent splicing activation, signifying novel targets in the fight against cancer [28]. It should be pointed out that abrogation of SOS under stress and in cancer might lead to profound dysregulation of the transcriptome, affecting a large variety of cellular functions, emphasizing transcriptomic instability in addition to the well-known genomic one. Therefore, targeting the unexplored thousands of LSSs that are activated in cancer and encode damaged proteins, having potentially harmful effects on cell metabolism, might lead to novel avenues in cancer diagnostics and treatment.

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